The overall goal of this research program is to characterize the mechanisms by which anti-tumor drugs that target DNA topoisomerases kill cells, to understand the biochemicals interactions between anti-tumor drugs and DNA topoisomerases, and to examine mechanisms that lead to drug resistance. We have developed a system using yeast as a tool to understand the actions of drugs that target DNA topoisomerases. Limited information exists on how a diverse range of compounds are able to inhibit DNA topoisomerase II in a similar biochemical fashion; by stabilizing the cleavable complex, we plan to examine the action of anti-topoisomerase by constructing mutants that are resistant to antitopoisomerase agents. The genetic tools available with yeast make it straightforward to isolate mutants within the topoisomerase II gene with specific phenotypes. We plan to concentrate on mutants that are resistant only to specific classes of drugs; this will help to exclude mutants that are drug resistant due to low enzyme activity. Once mutations are isolated with specific biochemical phenotypes, we will overexpress and purify the mutant proteins. This will enable us to characterize the changes that result in altered drug sensitivity. In addition to selecting for mutants that are drug resistant, we also plan to isolate mutants in topoisomerase II that are altered in relegation of the strand break that the enzyme introduces during its normal reaction. This type of mutant may be useful in understanding the details of drug inhibition. We also plan to take advantage of some new classes of drugs that may affect topoisomerase II differently than currently used clinical agents such as etoposide and mitoxantrone. Agents such as terpenticin may represent new , clinically useful topoisomerase II inhibitors; their unique biochemical effects on the enzyme represent another useful probe for understanding the action of anti-topoisomerase drugs. While the yeast gene is very homologous to human topoisomerase II, there may be some differences between human and yeast enzymes. We will construct a system that allows the functional expression of human topoisomerase II in yeast, and determine whether mutations in homologous positions produce similar changes in the yeast and human proteins. A variety of different mechanisms are likely to play a role in resistance to anti-topoisomerase drugs. For example, since the level of topoisomerase II is critical for sensitivity to drugs that target the enzyme, factors that alter topoisomerase II expression may alter drug sensitivity. Furthermore, many anti-topoisomerase II agents are substrates for MDR1, a protein that is able to transport cytotoxic agents-out of the cell. We propose to use the tools available in yeast to identify genes that play roles in sensitivity or resistance to anti-topoisomerase II agents.